Method for flaking chromosomes of phoebe nigrum
By using pretreatment, fixation, dissociation and chromatography methods during chromosome production, the problem of chromosomes prone to overlap in black shell nanmu is solved, and clear chromosome images are obtained, which significantly improves the breeding efficiency of black shell nanmu. and effects.
Patent Information
- Application Number
- CN202310949862.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-31
- Publication Date
- 2024-12-20
AI Technical Summary
During the chromosome production process, the chromosomes of Black Celestial Nano are prone to overlap and are not easy to obtain clear split phases, which leads to difficulties in breeding of Black Celestial Nano.
A method including pretreatment, fixation, dissociation and staining tableting is adopted: the material is pretreated at 3-5°C for 12-30 hours, fixed with Kano fixation solution and rinsed with 70%-95% ethanol. Then dissociate in 0.5-1 mol/L hydrochloric acid solution for 7-9 minutes, and finally staining tablets were performed on the slide for 7-9 minutes. The preferred material is the root tip of the main or lateral root of the seedlings of 15-20 cm long. The material extraction time is 9-11 am, the pretreatment time is 24 hours, the dissociation time is 8 minutes, and the dyeing time is 8 minutes.
A large number of split phases, well-dispersed and clear chromosomal images were obtained, which significantly improved the breeding value of Black Shell Nana.
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Figure CN119164739A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant chromosome preparation methods, and in particular to a method for preparing chromosomes of Machilus thunbergii. Background Art
[0002] Black shell nanmu belongs to the genus Piper in the Lauraceae family and is an evergreen broad-leaved tree species. Black shell nanmu has a beautiful shape and strong resistance, and is an excellent evergreen broad-leaved tree species that can be introduced and cultivated in northern China. Black shell nanmu also has rich volatile substances that can resist insects, bacteria and pollution, and its roots also contain anti-cancer drugs such as bicuculline, but the promotion and application of black shell nanmu in Henan Province is very lacking.
[0003] Plant chromosome preparation is the basis for cytogenetic studies such as karyotype analysis, banding analysis and chromosome in situ hybridization. In recent years, with the application of cytogenetic research in plant classification and breeding, chromosome preparation technology has become particularly important. However, in the current chromosome preparation process, chromosomes are prone to overlap when pressing, and it is difficult to obtain a clear division phase, making the breeding of black shell nanmu difficult. Summary of the invention
[0004] In order to solve the problem that chromosomes are easily overlapped and clear division phases are not easy to obtain during pressing in the prior art, making the breeding of Phoebe macrantho difficult, the present invention provides a method for preparing chromosomes of Phoebe macrantho.
[0005] A method for preparing chromosomes of Phoebe nigromaculata comprises the following steps:
[0006] (1) Pretreatment: The material for tableting is pretreated at 3-5°C for 12-30h;
[0007] (2) Fixation: The pretreated material is transferred to Carnoy's fixative and fixed at 3-5°C for 22-24 hours. The material is then rinsed with 70%-95% ethanol and stored for later use.
[0008] (3) Dissociation: After rinsing the fixed material, transfer it to a 0.5-1 mol / L hydrochloric acid solution and dissociate it at 55-65°C for 7-9 min;
[0009] (4) Staining and tableting: Stain the dissociated material for 7-9 minutes and press it into tablets.
[0010] Preferably, the material for making slices is the taproot or lateral root of a seedling of Phoebe macranthoides grown to 15-20 cm.
[0011] Preferably, the material for slice making is 0.5-1 cm from the root tip of the main root or lateral root.
[0012] Preferably, the material for film making is collected between 9 and 11 pm.
[0013] Preferably, the material is placed in an ice-water mixture and pretreated at 3-5° C. for 12-30 h.
[0014] Preferably, the pretreatment time is 24 hours.
[0015] Preferably, the Carnoy's fixative is prepared by mixing anhydrous ethanol and glacial acetic acid in a volume ratio of 2-4:1.
[0016] Preferably, after being rinsed with 95% by volume ethanol, the mixture is transferred into 70% by volume ethanol and stored at 4° C. for future use.
[0017] Preferably, the dissociation is carried out in a 1 mol / L hydrochloric acid solution at a temperature of 60° C. for 8 min.
[0018] Preferably, when staining and pressing, the non-meristem part is removed, leaving the white part of the root tip, placing it on a glass slide, and crushing the root tip.
[0019] Compared with the prior art, the present invention has the following beneficial effects:
[0020] The meristem obtained after the treatment of the present invention is used as a chromosome preparation material, and more mitotic phases, well-dispersed and clear chromosome images can be obtained, which is very valuable for the breeding work of Phoebe nanmu. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The results of chromosome preparation of Phoebe macranthoides seedlings, where (A) is the main root tip section, (B) is the lateral root tip section;
[0022] Figure 2 These are the chromosomes of the root tip cells of Pseudostellaria macranthoides at different division periods, among which (A), (B), and (C) are all at 8:00, (D) is at 10:00, (E) is at 12:00, and (F) is at 14:00;
[0023] Figure 3 The effect diagram of different pretreatment time of the root tip of Phoebe macranthoides, including (A) pretreatment for 2h, (B) pretreatment for 24h, (C) pretreatment for 48h;
[0024] Figure 4 The results of different dissociation time of the root tip of Phoebe macranthoides, including (A) dissociation for 6 minutes, (B) dissociation for 8 minutes, and (C) dissociation for 10 minutes;
[0025] Figure 5 These are the effects of different dyeing time on the root tip of Phoebe macranthoides, where (A) is dyed for 6 minutes, (B) is dyed for 8 minutes, and (C) is dyed for 10 minutes. DETAILED DESCRIPTION
[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified.
[0027] The black shell nanmu used was from Nanyang Xixia Seedling Station. After the seeds were collected, they were sown and raised in laboratory containers, and the test began when the seedlings grew to 15-20cm.
[0028] Instrument information for microscopy: Brand: Motic, Model: PA53 FS6 upright fluorescence microscope.
[0029] When examining under the microscope, first use the 10x microscope to find the field of view, then change to the 40x microscope, and observe the dispersion in a certain order along the 40x lower edge. When you find a cell field with better dispersion, drop oil and change to the 100x oil microscope. Select automatic exposure to take pictures, and then add a ruler uniformly.
[0030] Example 1
[0031] A method for preparing chromosomes of Phoebe nigromaculata comprises the following steps:
[0032] (1) Material collection: At 10 a.m., collect the taproot tip 0.8 cm from the 18 cm long seedlings of Phoebe macranthoides and wash them.
[0033] (2) Pretreatment: Place the obtained root apical material in a centrifuge tube filled with distilled water, place the centrifuge tube in a flask containing both ice and water, and place the flask in a refrigerator at 4°C for 24 hours before fixation for use.
[0034] (3) Fixation: The pretreated material was washed with distilled water, and Carnoy's fixative (glacial acetic acid: ethanol = 1 mL: 2 mL) was added and fixed at 4°C for 24 h. After fixation, the material was rinsed twice with 95% ethanol and then transferred to 70% ethanol and stored at 4°C for later use.
[0035] (4) Dissociation: The fixed material was rinsed with distilled water, transferred to a 1 mol / L hydrochloric acid solution, and dissociated in a 60°C water bath for 8 min.
[0036] (5) Staining and pressing: Wash the dissociated root tip in distilled water for 10 minutes, then remove the non-meristem part, keep the milky white root tip, place it on a slide, crush it with a dissecting needle, drop a drop (100 μL) of Carbofuchsin staining solution on each slide, stain for 8 minutes, cover with a cover glass and press it. For microscopic examination.
[0037] When pressing the slide, place the cover glass on the left side against the slide not far from the material, cover it with the staining solution, hold the cover glass with tweezers in your right hand and slowly lower it to expel the bubbles. Cover the cover glass with a piece of filter paper, fix the cover glass with your left hand, hold the eraser of a pencil in your right hand, aim at the root apex and press lightly to make a slice at a time.
[0038] Example 2
[0039] A method for preparing chromosomes of Phoebe nigromaculata comprises the following steps:
[0040] (1) Material collection: At 9:00 a.m., collect 0.5 cm of the taproot tip of a 15 cm long black-shelled Machilus macholii seedling and wash it.
[0041] (2) Pretreatment: Place the obtained root apical material in a centrifuge tube filled with distilled water, place the centrifuge tube in a flask containing both ice and water, and place the flask in a refrigerator at 3°C for 12 hours before fixation for use.
[0042] (3) Fixation: The pretreated material was washed with distilled water, and Carnoy's fixative (glacial acetic acid: ethanol = 1 mL: 3 mL) was added for fixation at 3°C for 22 hours. After fixation, the material was rinsed twice with 95% ethanol, and then transferred to 70% ethanol and stored at 4°C for later use.
[0043] (4) Dissociation: The fixed material was rinsed with distilled water, transferred to a 0.5 mol / L hydrochloric acid solution, and dissociated in a 55°C water bath for 7 min.
[0044] (5) Staining and pressing: Wash the dissociated root tip in distilled water for 10 minutes, then remove the non-meristem part, keep the milky white root tip, place it on a slide, crush it with a dissecting needle, drop a drop (100 μL) of Carbofuchsin staining solution on each slide, stain for 7 minutes, cover with a cover glass and press it. For microscopic examination.
[0045] When pressing the slide, place the cover glass on the left side against the slide not far from the material, cover it with the staining solution, hold the cover glass with tweezers in your right hand and slowly lower it to expel the bubbles. Cover the cover glass with a piece of filter paper, fix the cover glass with your left hand, hold the eraser of a pencil in your right hand, aim at the root apex and press lightly to make a slice at a time.
[0046] Example 3
[0047] A method for preparing chromosomes of Phoebe nigromaculata comprises the following steps:
[0048] (1) Material collection: At 11:00 a.m., collect 1 cm of the taproot tip of a 20 cm long black-shelled Machilus macholii seedling and wash it.
[0049] (2) Pretreatment: Place the obtained root apical material in a centrifuge tube filled with distilled water, place the centrifuge tube in a flask containing both ice and water, place the flask in a refrigerator at 5°C for 30 hours, and then fix it for use.
[0050] (3) Fixation: The pretreated material was washed with distilled water, and Carnoy's fixative (glacial acetic acid: ethanol = 1 mL: 4 mL) was added and fixed at 5°C for 23 hours. After fixation, the material was rinsed twice with 95% ethanol and then transferred to 70% ethanol and stored at 4°C for later use.
[0051] (4) Dissociation: The fixed material was rinsed with distilled water, transferred to a 1 mol / L hydrochloric acid solution, and dissociated in a 65°C water bath for 9 min.
[0052] (5) Staining and pressing: Wash the dissociated root tip in distilled water for 10 minutes, then remove the non-meristem part, keep the milky white root tip, place it on a slide, crush it with a dissecting needle, drop a drop (100 μL) of Carbofuchsin staining solution on each slide, stain for 9 minutes, cover with a cover glass and press it. For microscopic examination.
[0053] When pressing the slide, place the cover glass on the left side against the slide not far from the material, cover it with the staining solution, hold the cover glass with tweezers in your right hand and slowly lower it to expel the bubbles. Cover the cover glass with a piece of filter paper, fix the cover glass with your left hand, hold the eraser of a pencil in your right hand, aim at the root apex and press lightly to make a slice at a time.
[0054] The following experiments were performed to verify the effect of the method for preparing chromosomes of Phoebe sphenanthera of the present invention:
[0055] (1) Influence of different sampling sites
[0056] The taproots and lateral roots in the root tips of one-year-old seedlings of Phoebe macranthoides and the taproots and lateral roots of newly sprouted seedlings (seedlings grew to 15-20 cm) were selected for analysis.
[0057] The role of different sampling sites is to select the site with the most vigorous mitosis. The root tip is the site with the most vigorous mitosis. The results show that the main root and lateral root of the one-year-old seedling are thicker, which makes it more difficult to prepare the slides. In addition, the number of cells in the division stage of the meristem is very small, while the number of cells in the division stage of the main root and lateral root of the newly germinated seedling is very large, which greatly reduces the workload of microscopic examination. The results of chromosome preparation of the root tip cells of the main root and lateral root of the newly germinated seedling of P. maculatus are shown in Tables 1 and Figure 1 .
[0058] The results showed that most of the root tip cells of the taproot did not divide vigorously, with a large number of cells in the interphase and a small number of cells in the cell division phase ( Figure 1Figure A in the figure). The cells in the lateral roots are obviously in the cell division phase. The root tip of Phoebe macranthoides has the best preparation effect. The root tip cells in this part divide more vigorously, and there are many cells in the metaphase of division ( Figure 1 Figure B in the figure).
[0059] Table 1 Effect of sampling location on chromosome preparation
[0060]
[0061] Note: The number of “+” represents the degree of cell division.
[0062] (2) The impact of different sampling times
[0063] The purpose of selecting different sampling times is to select the time period with the most active mitosis, so as to facilitate the locking and acquisition of cells in metaphase for observation. The results of chromosome preparation of root tip cells of Phoebe maculatum at different sampling times are shown in Tables 2 and Figure 2 .
[0064] The results showed that most of the root tip cells sampled around 8:00 did not divide vigorously, and some of the chromosomes were dot-shaped (see Figure 2 A in Figure ), some are filamentous (see Figure 2 Some are tangled together, making it difficult to count (see Figure 2 Most of the root tip cells sampled at around 12:00 and 14:00 were entangled and had unclear outlines (see Figure 3). Figure 2 Figure E in Figure 2 Therefore, the best time to prepare the slices of the root tips of Phoebe maculatum is around 10:00. At this time, the root tip cells divide more vigorously, with many cells in metaphase, and the chromosome background is also clear and distinct (see Figure 2 Figure D in the figure).
[0065] Table 2 Effect of sampling time on chromosome preparation
[0066]
[0067] Note: The number of “+” represents the degree of cell division.
[0068] (3) Effect of different pretreatment times
[0069] There are two main effects of different pretreatment methods. One is to obtain more cells in metaphase, and the other is to further induce chromosomes to shorten and straighten, making chromosomes more dispersed and the staining morphology clearer.
[0070] The results of preparing slices of the root tips of Phoebe macranthoides with different pretreatment times are as follows: Figure 3It can be clearly seen from the figure that the pretreatment time has an impact on the dispersion and clarity of chromosomes in the root tip cells of Phoebe maculatum. When the pretreatment time is too short (2h), the chromosomes are not formed, most of them have not formed clear boundaries, and most of the chromosomes are stuck together. When the pretreatment time is too long (48h), the chromosomes in the root tip of Phoebe maculatum are mostly stuck together and blurred. The chromosomes in the root tip of Phoebe maculatum that are pretreated for 24h are more dispersed and the background is clear, which is conducive to the determination of chromosome number and karyotype analysis.
[0071] (4) Effect of different dissociation times
[0072] The purpose of dissociation is to separate the cells. After the cells are dispersed, it is easier to observe the number of chromosomes in a single cell. If the dissociation time is too long, the cells will rupture and the chromosomes will flow out. If the cell dissociation time is too short, the cells will overlap and it will not be convenient for microscopic observation.
[0073] Effects of different dissociation times on the root tip cells of Phoebe macranthoides Figure 4 As shown, the root tip cells dissociated for 6 minutes have unclear overlap, which is not conducive to the observation and counting of chromosomes; the root tip cells dissociated for 8 minutes have clear edges and no overlap, which is convenient for observing chromosomes and performing karyotype analysis; while the root tip cells dissociated for 10 minutes basically have no cell morphology, the dissociation is excessive, and the cells and chromosomes are scattered everywhere. The preparation effect of the root tip of the black shell macaw dissociated for 8 minutes is the most ideal.
[0074] (5) Effect of different dyeing times
[0075] If the staining time is too long, the cells and the degree of staining will be poor, and the cytoplasm will also be stained. If the staining time is too short, the staining will not be clear and is not conducive to observation.
[0076] The results of different staining times are as follows Figure 5 As shown in the figure, the root tip cells of the black shell macula dyed for 6 minutes are not deeply colored, which is not conducive to the analysis of chromosome morphology and number; the root tip cells of the black shell macula dyed for 8 minutes are well colored, and the chromosome morphology and number can be clearly observed; the root tip cells of the black shell macula dyed for 10 minutes are too darkly colored, which is not conducive to observation. Therefore, the root tip of the black shell macula dyed for 8 minutes has the best preparation effect.
[0077] The above experiments show that:
[0078] Ice bath shortens chromosomes, disperses chromosomes and has clear staining morphology. The root tip cells of newly germinated seedling lateral roots divide more vigorously, and the preparation effect is good. The root tip cells divide more vigorously when the material is collected at 10 am, and there are many cells in the metaphase of division. The background of chromosomes is also very clear and the morphology is distinct. The chromosomes of the root tip of the black shell nanmu pretreated for 24 hours are more dispersed and the background is clear, which is conducive to the determination of chromosome number and karyotype analysis. The edge of the root tip cells dissociated for 8 minutes is clear and there is no overlap, which is convenient for observing chromosomes and performing karyotype analysis. The root tip cells of the black shell nanmu root stained for 8 minutes are well colored, and the morphology and number of chromosomes can be clearly observed.
[0079] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.
[0080] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.
[0081] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.
Claims
1. A method for preparing chromosomes of Phoebe nigromaculata, characterized in that: The following steps are involved: (1) Pretreatment: The material for tableting is pretreated at 3-5°C for 12-30h; (2) Fixation: The pretreated material is transferred to Carnoy's fixative and fixed at 3-5°C for 22-24 hours. The material is then rinsed with 70%-95% ethanol and stored for later use. (3) Dissociation: After rinsing the fixed material, transfer it to a 0.5-1 mol / L hydrochloric acid solution and dissociate it at 55-65°C for 7-9 min; (4) Staining and tableting: Stain the dissociated material for 7-9 minutes and press it into tablets.
2. The method for preparing chromosomes of Pseudostellaria scabra according to claim 1, wherein: In step (1), the material for slice making is the taproot or lateral root of a seedling of Phoebe macranthoides grown to 15-20 cm.
3. The method for preparing chromosomes of Phoebe maculatum according to claim 2, wherein: The material for slice making is 0.5-1 cm from the root tip of the main root or lateral root.
4. The method for preparing chromosomes of Pseudostellaria scabra according to claim 3, characterized in that: The material for film making is collected between 9 and 11 am.
5. The method for preparing chromosomes of Pseudostellaria scabra according to claim 1, characterized in that: In step (1), the pretreatment is specifically to place the material in an ice-water mixture and treat it at 3-5°C for 12-30 hours.
6. The method for preparing chromosomes of Pseudostellaria scabra according to claim 5, characterized in that: The pretreatment time is 24 hours.
7. The method for preparing chromosomes of Pseudostellaria scabra according to claim 1, characterized in that: The Carnoy fixative is prepared by mixing anhydrous ethanol and glacial acetic acid in a volume ratio of 2-4:
1.
8. The method for preparing chromosomes of Pseudostellaria scabra according to claim 1, characterized in that: During the ethanol washing in step (2), after washing with 95% by volume ethanol, the mixture is transferred to 70% by volume ethanol and stored at 4° C. for later use.
9. The method for preparing chromosomes of Pseudostellaria scabra according to claim 1, characterized in that: In step (3), the dissociation is specifically carried out in a 1 mol / L hydrochloric acid solution at a temperature of 60° C. for 8 min.
10. The method for preparing chromosomes of Pseudostellaria scabra according to claim 1, characterized in that: In step (4), when staining and pressing, remove the non-meristem part, keep the white part of the root tip, place it on a slide, and crush the root tip.